The Pharmacological Effects of Odonthobuthus doriae Scorpion Venom and Its Extracted Fractions on Neuro-Muscular Transmission

The effect of Odonthobuthos doriae (O.d) scorpion venom at 0/3, 1 and 3, 10 µg/ml concentrations were investigated on nerve-muscle transmission, using the Twitch tension technique. A concentration of 0.3 µg/ml caused a small change in the twitch height in response to indirect muscle stimulation, but higher concentrations (1, 3, 10 µg/ml) caused a transient augmentation in twitch response followed by a large contracture in the chick biventer cervices (CBC) preparation. This effect could be defined as a complex action of the venom, predominately presynaptic, in which its’ effects on postjunctional synapses is also maintained. In order to find out which bioactive fraction could explain the venom effects, the soluble crude venom was partially separated by the gel filtration method, using a Sephadex G50 column, and four fractions were separated. Two of the four purified fractions (O.d F3, O.d F4) were characterized as toxic and their LD50 values were lower than the crude venom. Unlike the O.d F1 and O.d F2 fractions, O.d F3 and O.d F4 fractions caused a significant block in the twitch and contracture, in comparison to the control sample . in conclusion, fractions O.d F3 and O.d F4 are supposed to be as the biological active components of the O.d. venom.

The effect of Odonthobuthos doriae
(O.d) scorpion venom at 0/3, 1 and 3, 10 ?g/ml concentrations were investigated
on nerve-muscle transmission, using the Twitch tension technique. A
concentration of 0.3 ?g/ml caused a small change in the twitch height in
response to indirect muscle stimulation, but higher concentrations (1, 3, 10
?g/ml) caused a transient augmentation in twitch response followed by a large
contracture in the chick biventer cervices (CBC) preparation. This effect could
be defined as a complex action of the venom, predominately presynaptic, in
which its? effects on postjunctional synapses is also maintained.

?In order to find out which bioactive
fraction could explain the venom effects, the soluble crude venom was partially
separated by the gel filtration method, using a Sephadex G50 column,
and four fractions were separated. Two of the four purified fractions (O.d F3,
O.d F4) were characterized as toxic and their LD50 values
were lower than the crude venom. Unlike the O.d F1 and O.d F2
fractions,
O.d F3 and O.d F4 fractions caused a significant block in
the twitch and contracture, in comparison to the control sample . in
conclusion, fractions O.d F3 and O.d F4 are supposed to
be as the biological active components of the O.d. venom.

Scorpion toxins
have had instrumental effects in defining both structural and mechanistic
properties of ion channels and various conductances normally present in cells
involved in its blocking activity (1).

Up to now, more than 1500 genus of
scorpions are known word wide, and the most hazardous scorpions to human are
those belonging to the Buthidae family. Odonthobuthus belongsto
the Buthidae family and Buthus genus which has two species, doriae
and odonthurus. This scorpion has an abundant geographical distribution
in Iran. Its? sting can cause various effects ranging from pain, inflammation,
and necrosis at the site of sting, to muscle paralysis, hematuria and
occasionally death in children.

Pharmacological characterizations of
this venom, as well as its? sub-fractions has not yet been reported. In this
study, the in vitro pharmacological effects of the venom and its? toxic
fractions extracted by gel filtration have been investigated on the
neuromuscular transmission, using the Chick Biventer Crevices (CBC)
preparation.

Experimental

Materials

Crude scorpion venom was gifted by
Dr. Zare and Dr. Akbary, Department of Poisonous animal, Vaccine and Serum
Research Institute of Razi, Karaj, Iran. Crude venom was obtained by electrical
stimulating telson of scorpion and stored after freeze-drying at ?50?C, until
use.

Twitch tension recording experiments
were performed on the isolated CBC nerve-muscle preparation. This model is
suitable for studying the nerve-muscle transmission effects of drugs or venom.
It is small enough to be mounted within a 2-5 ml tissue organ bath and retain
its? normal function in vitro, over a few hours.

?

Chick biventer cervices preparations

Biventer cervices
muscles and associated nerves were dissected from 7-10 days old chicks,
sacrificed by exposure to ether as described by Ginsborg and Warriner (2). Two
muscles were mounted, with a resting tension of approximately 1 g; within a 5
ml tissue bath containing modified physiological solution (mM) as follows:
NaCl, 118.4; KH2PO4, 1.2; Glucose, 11.1; NaHCO3,
25; CaCl2, 2.5; MgSO4, 1.4 and KCl, 4.7. Twitches were
evoked by stimulating the motor nerve at 0.1 Hz with pulses of 0.2 ms duration
and a voltage greater than that which produced a maximal response, using ring
electrodes and a Grass S88 B stimulator. To detect any changes in postsynaptic
sensitivity, responses to sub-maximal concentrations of exogenously applied
acetylcholine (1-2 mM), carbachol (30-40 μM) and KCl (20-40 mM) were
recorded in the absence of nerve stimulation, prior to the addition of venom
and at the end of the experiment. The preparations were exposed to
acetylcholine and KCl for 30 sec, and carbachol for 1 min (3). After the wash-out
stage of these drugs, the preparations were allowed to stabilize for 15-20 min
before the addition of the venom.

Twitches and contractures were
recorded isometrically, using the Washington Grass model 79B and Grass model 79
polygraphs and SRI or Grass FT03 force transducers.

Purification Procedure

Fractioning of the soluble venom was
accomplished using a Sephadex G50 column (3.5?230 cm, 1 ml/min flow)
equilibrated and eluted with a pH 8.3 0.1 M ammonium acetate buffer, as
previously described (4). Protein content was estimated spectrophotometrically
at 215, 260 and 280 nm. All the procedures were carried out at room
temperature.

Lethality tests and LD50
determination in mice

The mice (white, of both sexes,
weighing 20 g) used in this research were treated in compliance with the US
Public Health Service policy on human care and the use of animals. The mouse
lethality of various protein fractions was observed after i.v. injections to
mice (5). The Reed and Muench method, which provides a moderately accurate estimate
of the LD50 based upon data smoothened by counting cumulative number
of survivals, was used (6). Two designations of ?toxic? and ?nontoxic?? were
used to express venom fractions. A fraction or peptide was considered
non-toxic, if during the first 24 h of its injection the mouse lived or showed
no more toxicity signs (heperexcitability, lachrymation, apnea, partial
paralysis or respiratory failure). Toxic fraction means a fraction that
immediately caused death of the test animal. The control group received an
injection of normal saline at pH 7.4.

Data analysis was performed using the
one-way (repeated) analysis of variance (ANOVA) for multiple comparison and
student?s t-ttest for comparing two set of data as Mean ? SEM (p<0.05).

Results
and Discussion

Effects of the venom on chick
biventer cervices preparations

Figure 1 shows that the twitch
responses of CBC evoked by nerve stimulation were reduced in the presence of Odonthobuthus
scorpion venom (0.3-10 μg/ml), in a time-dependent manner. In contrast to
other concentrations, the effect of 10 μg/ml venom was extremly strong and
without any transient increase in the twitch height. It blocked muscle
twitching within 13 min. As could be seen in figure 1, there was a direct
relation ship between concentration and the maximum contracture effect.

With 3 and 10 μg/ml
concentrations, the effect of venom was much faster and stronger in the
indirectly stimulated preparation than the direct preparation (Figure 2).

Effects of
the venom on acetylcholine, carbachol and potassium chloride responses, without
electrical stimulation

Figure 3 shows a significant (P<0.05) decrease
of Acetylcholine (1-2 mM) for 30 sec, Carbachol (30-40 ?M) for 1 min and KCl
(30-40 mM) for 30 sec CBC contracture responses, after exposure to 1, 3, and 10
?g/ml concentrations of the venom. Meanwhile, the responses to a concentration
of 0.3 ?g/ml of venom in acetylcholine and carbachol was not significant. As
shown in Figure 3, the decreasing pattern of acetylcholine and potassium
chloride is similar at different concentrations of venom.

Figure 4 shows
a gradually significant reduction in the twitch height responses of CBC
preparations stimulated indirectly at different concentrations of 1, 3, and 10
?g/ml of O.d F3, which was followed by complete blockage.

The effect
of O.d F4 fraction on twitch height in indirect electrical
stimulation

Figure 5 shows
a gradually significant reduction in twitch height responses of CBC
preparations stimulated indirectly at different concentrations of 1, 3, and 10
?g/ml of O.d F4, in comparison to the control, leading to muscle
paralysis at 1 and 10 ?g/ml concentrations. A complete blockage at 1 and 10
?g/ml concentrations was observed after 21 and 18 min, respectively.

Skeletal muscle
paralysis could occur due to either a presynaptic action via
blockade/acceleration of acetylcholine release, or to a postsynaptic effect
through blockage of acetylcholine receptors or direct muscle damage. In this
study, experiments were performed by the indirect, direct and without
electrical stimulation of CBC preparation, in order to investigate the pre- or
post-synaptic effects of the O.d venom and its? toxic fractions.

According to the data obtained, it seems that the
effects of the venbom are mostly mediated through presynaptic effects on nerve
terminals. Our results are in agreement with the Pandinus imperator
scorpion effects on CBC preparation (7). Furthermore, the same effects were
noted earlier with Pandinus exitialis and Tityus serrulatus
scorpion venoms on mouse hemi-diaphragm preparation (8, 9).

At higher
concentrations, the inhibitory responses of CBC to Ach and Carb were boosted.
This effect may be due to the blockage of neuro-muscular transmission, arising
from the effects of the venom on nicotinic receptors of the postsynaptic
membrane (10). The inhibitory effects of venom in response to Ach and Carb,
suggest the curar?mimetic effects of venom. However, the contracture response
observed as a result of direct stimulation, after the addition of
d-Tubocurarine, can rule out this suggestion (11). Further more, the
significant reduction in response to KCl at a venom concentration of 0.3 ?g/ml,
contributes to the direct effect of venom on muscle (12).

The influence
of 1, 3 and 10 ?g/ml concentrations of two non-toxic fractions, O.d F1
and O.d F2, were also studied on CBC preparations (table 1). There
were no significant pharmacological changes in response to indirect
stimulation. In contrast, O.d F3 and O.d F4 fractions result in significant
changes in twitch height and yield in a complete blockage. Comparison of
different concentrations of O.d F3 and Od F4, show a similar potency in
paralysis of twitch tension responses (64% and 63%, respectively).

Finally,
according to our obtained data, O.d venom acts mostly prejunctionally to
inhibit the neuromuscular transmission. Further studies to find out which
bioactive molecule is responsible for the venom action, showed that O.d F3
and O.d F4 fractions, which have a similar potency, are the active
components of this venom. These findings may warrant research into the
development of purification and extraction of a specific pharmacological agent
against channels, from the Odonthobuthus venom.

7.
Mashall DL and Harvey AL. Block of potassium channels and
facilitation of acetylcholine release at the neuromuscular junction by the
venom of the scorpion Pandinus imperator. Toxicon (1989) 27:
493-8